This proposal remains focused on applications of novel fluorescence methods to measure solute mobility and interactions in living cells and tissues. The central goal of this research is to utilize information about solute dynamics to generate a realistic picture of the intracellular milieu and tissue extracellular spaces that cannot be deduced from static measurements such as electron micrographs. The current proposal utilizes new methodologies (microfiberoptic photobleaching, quantum dot single particle tracking, two-color and polarization fluorescence cross-correlation microscopies) to study molecular diffusion in cell culture and in vivo (mouse) systems. Aim 1 will measure diffusion in the extracellular space (ECS) of tumor and brain tissues to test the hypothesis that ECS matrix composition and tortuosity restrict macromolecule diffusion. A microfiberoptic epifluorescence photobleaching (MFEP) method will be used to measure diffusion deep in tumor and brain tissue in vivo to quantify the contributions of extracellular matrix composition vs. geometric factors. Aim 2 will measure plasma membrane diffusion of individual G-protein coupled receptors (GPCRs) to test the hypothesis that cytoskeletal and intrinsic membrane barriers restrict GPCR diffusion. High- resolution tracking of individual, quantum dot (QD)-labeled beta-2 and vasopressin-2 receptors, together with mutagenesis and pharmacological maneuvers, will be used to identify the determinants of receptor diffusion. Aim 3 will measure protein interactions and rotational dynamics by fluorescence cross-correlation spectroscopy to test the hypotheses that mitochondrial matrix enzymes and membrane CFTR chloride channels form multi-molecular complexes. Following on from extensive preliminary data about the biology of mitochondrial matrix enzymes of the tricarboxylic acid cycle, and CFTR chloride channels, we will use cross- correlation microscopy to define the degree of complexation of mitochondrial matrix enzymes, and role of C- terminus PDZ interactions in restricting CFTR rotational mobility.